This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Progress in molecular imaging of metabolic processes will depend heavily on the availability of new molecules as sensors. In this project, we focus on developing the chemistry to build new, strategically-designed molecules for molecular imaging of metabolite levels and pathway fluxes by MRI. In aim 1, our focus will be on new glucose derivatives for hyperpolarized 13C imaging of glycolysis and the pentose phosphate pathways. We propose a new strategy of storing 13C polarization in a single long T1 site then, as metabolism happens, transfer the polarization of other spin-coupled sites in the same molecule that are more characteristic of downstream metabolites. In this way, one can use the chemical shifts of shorter T1 protonated carbons to readout downstream metabolites. In aim 2, we will apply new hyperpolarized 13C sensors for imaging tissue redox and pH. Aim 3 focuses on development of a new type of enzyme-based nanosensor for molecular imaging of a specific biological target while aim 4 focuses on continued development of a PARACEST agent for imaging the extracellular distribution of glucose in animals. Aim 1 is to develop long-T1 glucose analogs for imaging tissue uptake and metabolism of glucose. We have already synthesized [1-13C]gluconolactone and shown that it is readily hyperpolarized, transported into cells by glucose transporters, phosphorylated, enters the pentose phosphate pathway (PPP) and yields HP[H13C03-] after two enzyme catalyzed steps. We will continue supplying [1-13C]gluconolactone to TR&D Project 2 and focus on developing new long T1 glucose derivatives for storing polarization long enough to allow metabolic processes to occur. Excess polarization will then be transferred to other spin-coupled carbons in the glucose molecule prior to an observation pulse to identify glucose-6-P and other possible downstream metabolites. Our goal is to create new systems that remain polarized for 3-5 minutes or longer. Aim 2 is to develop long-T1 redox and pH sensors for hyperpolarized imaging of abnormal physiology. Acid generation is a fundamental property of metabolism so having a convenient method for imaging tissue pH would be extremely valuable for identifying hypermetabolic tissues such as cancer and infection. Hematopoietic stem cells and progenitors express high levels of aldehyde dehydrogenase (ALDH1) and this has become a popular target for identifying and purifying these cells. We hypothesize that long T1 HP-13C-aldehyde-based probes will be useful for monitoring flux through ALDH1 as tumors respond to drug therapy. Aim 3 is to develop a family of nanoscale MRI detectors capable of imaging specific biological targets in vivo. MRI is considered too insensitive for molecular imaging of biological targets in vivo. This project will focus on development of single enzyme nanoparticles (SENs) for converting an enzyme-specific substrate having a long T1 carbon into specific product only in regions where SENs probes are targeted. This technology will allow imaging of specific molecular targets in vivo at a 20,OOO-fold or more sensitivity advantage over conventional MRI. Aim 4 is to image the extracellular distribution of glucose in vivo using a glucose-sensitive paramagnetic CEST agent and SWIFT imaging. Imaging the distribution of glucose using CEST principles has been a long-term goal but the glucose sensor reported previously was found to have two problems that limited success in that project. Those problems have now been identified and solved. The line broadening T2exch effects that limited detection of PARACEST agents in vivo previously are now circumvented by using of SWIFT imaging principles. This exciting advance will now allow imaging of responsive PARACEST agents in vivo, including the new glucose sensor. This technology achievement opens the door to imaging sensors of metabolism.